Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5’-UTR region

(1)

RESUMO

RESUMO.- [Caracterização genética de amostras brasileiras

Caracterização genética de amostras brasileiras

do vírus da Diarréia viral bovina através do seqüenciamento

parcial da Região 5’UTR

parcial da Região 5’UTR.] Dezenove amostras do vírus da

diar-réia viral bovina (BVDV) foram caracterizadas geneticamente

atra-vés do seqüenciamento parcial de nucleotídeos da Região 5’UTR.

As amostras foram agrupadas em BVDV-1 (11/19), BVDV-2 (6/19)

e num terceiro grupo de amostras denominadas “atípicas” (2/

19). Das onze amostras genotipadas como BVDV-1, oito

amos-tras foram sub-genotipadas como BVDV-1a, enquanto que a

maioria (4/6) das amostras de 2 foi agrupada como

BVDV-2b. Duas amostras provenientes de fetos bovinos abortados

fo-ram classificadas como atípicas, não BVDV-1 e 2. A presença da

diversidade genética de BVDV detectada nas amostras

estuda-das pode ser responsável por falhas vacinais e de diagnóstico e

deve influenciar nas estratégias de controle do BVDV aplicadas

nas diferentes regiões brasileiras.

TERMOS DE INDEXAÇÃO: BVDV-1, BVDV-2, Pestivirus, Brazil, análise filogenética.

INTRODUCTION

Infections of cattle by bovine viral diarrhea virus (BVDV) are

widespread cause of major economic losses to the cattle industry

(Houe 1999). Clinical symptoms may involve the reproductive,

respiratory, immune, and gastrointestinal systems, with signs that

may range from disease with high mortality rates to asymptomatic

infections. The latter is observed in most cases (Pellerin et al. 1994,

Ridpath et al.1994, Baker 1995, Fray et al. 2000).

BVDV is an enveloped RNA virus that belongs to family

Flaviviridae, genus

Pestivirus.

The viral positive single stranded

genome of approximately 12.5 kb in size contains a single open

reading frame (ORF) flanked by two non-translating terminal

regions, named 5’ and 3’-UTR. The single ORF is directly translated

and gives rise to a long polyprotein which is co-translationally

cleaved, originating 10 to 12 mature viral proteins (Collet et al.

1988, Meyer et al. 1989).

Isolates have been subdivided in genotypes 1 and

BVDV-2. These were further split into subgenotypes. To date, 11

1_{Received on June 8,2006.}

Accepted for publication on July 25, 2006.

2_{Faculdade de Medicina Veterinária e Zootecnia, Universidade de São}

Paulo, Av. Prof. Dr. Orlando de Paiva 87, Cidade Universitária, São Paulo, SP 05508-000, Brazil. *Corresponding author: leonardo@usp.br

3_{Escola de Veterinária, Universidade Federal de Minas Gerais, Cx.Postal}

567, Belo Horizonte, MG 30123-970.

4_{Instituto Biomédico, Universidade Estadual do Norte Fluminense Darcy}

Ribeiro, Av. Alberto Lamego 2000, Campos dos Goytacazes, RJ 28013-600.

5_{Centro de Ciências Agrárias, Universidade Estadual de Londrina, Cx.}

Postal 6001, Londrina, PR 86051-990.

6_{Centro de Ciências Rurais, Universidade Federal de Santa Maria,}

97105-900 Santa Maria, RS.

Genetic characterization of Brazilian bovine viral diarrhea virus

isolates by partial nucleotide sequencing of the 5’-UTR region

1

1 Adriana Cortez

2

_{, Marcos B. Heinemann}

3

_{, Alessandra Marnie M.G. de Castro}

2

_,

Rodrigo M. Soares

2

_{, Ana Maria V. Pinto}

4

_{, Amauri A. Alfieri}

5

_{, Eduardo F. Flores}

6

_,

Rômulo Cerqueira Leite

3

_{and Leonardo J. Richtzenhain}

2*

ABSTRACT

ABSTRACT.- Cortez A., Heinemann M.B., Castro A.M.M.G., Soares M.S, Pinto A.M.V., Alfieri A.A.,

Flores E.F., Leite R.C. & Richtzenhain L.J. 2006. Genetic characterization of Brazilian bovine

Genetic characterization of Brazilian bovine

viral diarrhea virus isolates by partial nucleotide sequencing of the 5’-UTR region

viral diarrhea virus isolates by partial nucleotide sequencing of the 5’-UTR region.

Pesquisa

Veterinária Brasileira 26(4):211-216.

Faculdade de Medicina Veterinária e Zootecnia, Universidade

de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitária, São Paulo, SP

05508-000, Brazil. E-mail: leonardo@usp.br

Nineteen isolates of bovine viral diarrhea virus (BVDV) from Brazil were genetically characterized

through partial nucleotide sequencing and analysis of the 5’UTR region. The isolates were grouped as

BVDV-1 (11/19), BVDV-2 (6/19) or “atypical” pestivirus (2/19). Among the BVDV-1, eight isolates were

classified as subgenotype BVDV-1a, whereas most (4 out of 6) BVDV-2 belonged to subgenotype 2b. Two

isolates from aborted fetuses were not classified into any genetic group, being considered atypical

BVDVs. Genetic diversity among Brazilian BVDV isolates may be responsible for vaccination and

diag-nostic failure and therefore may influence the control strategies for BVDV infection in the country.

(2)

subgenotypes of BVDV-1 (Vilcek et al. 2001) and 2 subgenotypes

of BVDV-2 (Flores et al. 2002, Vilcek et al. 2004) have been described.

Genotyping and subgenotyping of BVDV isolates have been

accomplished mainly by the analysis of 5’-UTR region, since it is

highly conserved among pestiviruses (Ridpath et al. 1994,

Sandvick et al. 1997, Ridpath & Bolin 1998, Letellier et al. 1999,

Baule 2000, Flores et al. 2002). The aim of the present study was

to characterize bovine viral diarrhea virus isolates from cattle

affected by different clinical syndromes in Brazil by partial

nucleotide sequencing of the 5’-UTR region.

MA

MATERIALS AND METHODS

TERIALS AND METHODS

Twelve BVDV field strains isolated in tissue culture (Flores et al. 2005) and seven BVDV field isolates directly detected by reverse transcription followed by PCR (RT-PCR) in clinical samples (Cortez et al. 2005b) were submitted to partial sequencing of 5’UTR region for genotyping and subgenotyping.

RNA extraction was performed using TRIzol reagent (Invitrogen). The RNA was reverse-transcribed with the Moloney Murine Leukemia Virus-Reverse Transcriptase using random primers (MMLV-RT; InvitrogenTM_{). Amplification of a 5'-UTR fragment was obtained with} the primers P1 and P2 described by Ridpath & Bolin (1998). A

hemi-nested PCR was carried out employing the primers P1 and PN2 (5’ RCA CCC TWW CAG GCT GT 3’) as internal primers.

Sequencing was carried out with primers P1 and PN2, using the Big Dye TM Terminator-cycle sequencing ready reaction kit (Applied Biosystems). Sequences were examined with the software PHRED (http:// bioinformatica.ucb.br/electro.html) for quality analysis of chromatogram readings. The sequences were accepted if their quality readings were equal to or higher than 20. The consensus sequences were determined by the software CAP3 (http://bioinformatics.iastate.edu/aat/ sas.html) and the alignment was obtained with the aid of the software BioEdit (Hall 1999). The sequences described in Table 1 were used for comparison purposes. The presence of a phylogenetic signal was verified by the saturation in the curve of transition versus transversion calculated by the software DAMBE (Xia & Xie 2001).

In order to determine the evolutionary model of nucleotide substitution for the group of sequences analyzed, likelyhood-ratio tests (Hueselsenbeck & Rannala 1987) were used with the aid of the software ModelTest (Posadas & Crandall 1988).

Utilizing the software Mega (Molecular Evolutionary Genetics Analysis) v.2.0 (Kumar et al. 2000), 55 taxa of 167 nucleotides, from a total of 358 nucleotides of 5’-UTR region, were analyzed by Neighbor-Joining (NJ) method. The values of the genetic distances and bootstraps were calculated. Bootstrap calculations were performed by the heuristic search method with 1000 copies.

T TT

TTable 1. Pable 1. Pable 1. Pable 1. Pable 1. Pestivirus isolates, indicating accession numberestivirus isolates, indicating accession numberestivirus isolates, indicating accession numberestivirus isolates, indicating accession number, reference, origin of the isolate andestivirus isolates, indicating accession number, reference, origin of the isolate and, reference, origin of the isolate and, reference, origin of the isolate and, reference, origin of the isolate and classification

classificationclassification classificationclassification

Isolates Gene Bank References Origin of strains BVDV Subgenotype

Access Number

EVI 006 Not available Gil 1998 Rio Grande do Sul/Brazil 1a

INTA 5 Not available Gil 1998 Argentina 1a

126/1 Not available Gil 1998 Rio Grande do Sul/Brazil 1a

NADL M31182 Collet et al. 1998 USA 1a

akt1 AY 443029 Jones et al. 2004 Argentina 1a

68.88 AF 244962 Jones et al. 2001 Argentina 1a

UFSM 1 Not available Gil 1998 Rio Grande do Sul/Brazil 1b

UFSM 2 Not available Gil 1998 Rio Grande do Sul/Brazil 1b

IBSP2 Not available Gil 1998 São Paulo/Brazil 1b

IBSP4 Not available Gil 1998 São Paulo/Brazil 1b

INTA 1 Not available Gil 1998 Argentina 1b

126/8 Not available Gil 1998 Rio Grande do Sul/Brazil 1b

Strain NY-1 L32879 Pellerin et al. 1994 USA 1b

3P AF244968 Jones et al. 2001 Argentina 1b

66.1 AF244953 Jones et al. 2001 Argentina 1b

SE 6530 1580751 GenBank Not informed 2a

97/730 AF026770 GenBank New Zealand 2a

SW-90 AB003622 Nagai et al. 1998 Japan 2a

Strain 890 U18059 Ridpath & Bolin 1995 USA 2a

167 237 U65055 Vicek et al. 1997 Wales 2a

1373 AF145967 Ridpath et al. 2006 USA 2a

VM 97 Not available Gil 1998 Rio Grande do Sul/Brazil 2b

SV63 Not available Gil 1998 Rio Grande do Sul/Brazil 2b

Soldan U94914 Canal et al. 1998 Rio Grande do Sul/Brazil 2b

34B AF 244952 Jones et al. 2001 Argentina 2b

D32_00 HoBi AY489116 Schirrmeier et al. 2004 Brazil Atypical pestivirus

Rudolph AB122086 GenBank Not informed Border Disease Virus (BDV)

Casimir AB122085 GenBank Not informed BDV

CSFV/MP AY182247 GenBank Not informed Classic Swine Fever (CSFV)

INB-03 DQ191444 GenBank India CSFV

unnamed AY306121 GenBank Not informed CSFV

(3)

Two-sample t Tests were carried out using the software MINITAB to compare the average genetic distances between each group of BVDV sequences. The sequences used for this analyze were grouped according to the results obtained with the phylogenetic analysis (Fig 1). The group BVDV-1 contains the average genetics distances of the sequences of genotype BVDV-1, the group BVDV-2, is formed by isolates belonging to the genotype BVDV-2, the Atypical Pestivirus holds the atypical pestivirus sequences. The External Group contains the sequences of Border Disease and Classical Swine Fever Virus. Values for p ≤_{0.05 were considered statistically significant.}

RESUL

RESULTS

TS

The analysis of the phylogenetic signal showed that saturation

between transition and transversion did not occur in the

sequences utilized, indicating that these may be used in

phylogenetic reconstruction.

The most appropriate evolutionary model of nucleotide

substitution for the group of sequences analyzed was the Kimura

two-parameter model.

Eleven isolates had their genotypes determined as BVDV-1

(eight as subgenotype 1a and three as subgenotype

BVDV-1b) and six as BVDV-2 (four subgenotypes BVDV-2b and two as

subgenotype BVDV-2a). Two isolates formed a group with the

D32/00_HoBi, an atypical pestivirus isolates from Brazilian foetal

bovine serum (Table 2, Fig.1).

The statistic analysis, using two-sample t Test, of the genetic

distances for the sequences grouped in BVDV-1, BVDV-2, Atypical

Pestivirus and External Group showed that only the average

values of BVDV-1 x BVDV-2 were not statistically significant,

meaning that the average values of the genetic distances of these

groups are not different. The average values between the other

groups BVDV-1 x Atypical Pestivirus, BVDV-1 x External Group,

BVDV-2 x Atypical Pestivirus , BVDV-2 x External Group, Atypical

Pestivirus x External Group were statistically significant (p<0.05).

It was not possible to establish any spatial, temporal and/or

symptomatic statistical correlation with specific genotypes or

subgenotypes (Table 2).

DISCUSSION

The genotypes and subgenotypes obtained in this study by partial

nucleotide sequencing of 5’-UTR region (Fig.1, Table 2) confirm

the results reported by other authors (Canal et al. 1998, Gil et al.

1998, Flores et al. 2002, 2005) that demonstrated the presence

of BVDV-1 and BVDV-2 in various Brazilian states. The finding of

subgenotypes BVDV-1a and BVDV-1b corroborates the data by

Gil (1998) and Gil et al. (1998). Although no other BVDV-1

subgenotypes have been evidenced in the present study, Vilcek

et al. (2004) described the existence of the subgenotype

BVDV-1d in Brazil.

The wide distribution of the clinical manifestations for the

two genotypes (Table 2) supports others studies that verified the

pathogenic and genetic diversity associated with Brazilian isolates

(Gil et al. 1998, Flores et al. 2002, 2005).

Within subgenotype BVDV-1a, the isolates 132, EVI006,

UFSM3 and 1/5 displayed 100% identity. This sequence was found

in isolates from the State of Rio Grande do Sul (EVI006) in the

90’s, and in the States of Minas Gerais (1/5) and Rio Grande do

Sul (132 and UFSM3) in 2004. In Paraná State, in 2003, a sister

group was observed (303). The isolate 3/4 from State of Mato

Grosso do Sul formed a sister-group with the isolates 303, 132,

UFSM3, 1/5, EVI006. The results suggest that viruses showing

this genetic profile are circulating in the regions South, Southeast

and Central-Western Brazil. The transmission between the States

could have occurred through of trade infected animals, of

cryopreserved vehicles like semen and embryos as well in

artifi-cial reproductive techniques (Lindberg & Houe 2005).

The most isolates (278, 3/4 303, UFMS3 and 1/5) formed a

cluster predominantly connected to reproductive manifestation

that could not be showed in the sister-group (216, 663; Fig.1,

Table 2) and was presented in the States of Rio Grande do Sul,

Paraná and Minas Gerais. Jones et al. (2004) suggested that isolates

linked to reproductive disorders are more recent than enteric

isolates.

The isolate IBSP4, obtained from clinical sample in 1995, in

Ribeirão Preto, SP, formed a distinct group in relation to the

others of subgenotype BVDV-1b, supported by a bootstrap value

higher than 70. In the same year, in the city of Jaboticabal, SP,

distant from Ribeirão Preto by 50 km, an isolate of the sister

group (IBSP2) was obtained, showing that, in the same year and

in herds located a few kilometers apart, different genetic types

of the virus may circulate concomitantly.

Still regarding the subgenotype BVDV-1b, in 1996, similar

nucleotide sequences were found in the isolates UFSM1, UFSM2,

126/14, 153/1, obtained from healthy bovine fetuses collected

in slaughterhouses in different cities in the State of Rio Grande

do Sul. In 2001, in Arroio dos Ratos, RS, a virus harboring a

similar sequence (163) was isolated from an animal presenting

respiratory and digestive signs. Different clinic manifestation in

the other cluster (390, 395) could also be demonstrated (Fig.1,

Table2).

Factors associated with host, the environment and agent can

influence the clinical outcome of BVDV (Baker 1995, Hamers et

al. 2001) and despite Jones et al. (2004) and Baule et al. (2001)

related to subgenotypes BVDV-1b and BVDV-1d with respiratory

disease, a correlation between BVDV genotypes and

subgeno-types and clinical signs has not yet been evidenced (Ripath et al.

1994, Lettelie et al. 1999, Fulton et al. 2005).

Among the genotype BVDV-2, two were identified as

subgenotype BVDV-2a and four as subgenotype BVDV-2b. Flores

et al. (2002) and Vilcek et al. (2004) suggested that there is a

possible geographic link between North America and

subgenotype BVDV-2a, and between South America and

subgenotype BVDV-2b.

In the present study, a wide range of clinic manifestation

associated with of BVDV-2 isolates was observed ( Table 2). There

were two subgroups of BVDV-2a circulating, the isolate 44

collected in State of São Paulo in 1997 clustered into same

subgroup with United State of America, Wales, Japan, New

Zealand isolates and a BVDV positive (323) gathered in 2004 that

was segregated in a distinct subgroup. It would be necessary a

larger number of isolates to verified if this subdivision correlates

with traits epidemiology like time of isolation.

(4)

(5)

se false-negative results in serologic diagnosis as the

serum-neutralization test (Flores et al. 2000).

The finding of two isolates (200 and 315) that grouped

together with D32/00_ HoBi isolate (Schirrmier et al. 2004) is

supported by phylogenetic reconstruction, by high bootstrap

values that separate the ramifications of the various groups (Fig.1)

and by statistic analysis, that shows that there is a significant

difference between the average values of the genetic distances

for group D32/00_HoBi and the other groups. The D32/00_

HoBi isolate was first described in Germany, in 2004, as a

contaminant of Brazilian fetal bovine serum and displays antigenic

and genetic characteristics that are distinct from other

Pestivirus.

These data suggest that these isolates may belong to a recently

described new species within the genus Pestivirus. In the animal

experiment, inoculated calves did not show clinical signs

(Schirrmier et al. 2004). The two isolates described in this study,

200 and 315, were obtained from bovine foetuses aborted in

2002 (200) in São Sebastião da Grama, and in 2004 (315) in

Tambaú, both cities located in the State of São Paulo, about 100

km apart from each other, a fact suggesting that these isolates

were probably circulating among cattle and causing reproductive

disorders in the region.

Despite the relatively small number of isolates analyzed, yet

the source of the isolates being mostly from Central-Southern

Brazil, the diversity observed among Brazilian BVDV isolates

obtained from different clinical syndromes associates the

antigenic differences of several genotypes, subgenotypes and a

presence of new species of Pestivirus causing reproductive

disorders may contribute to diagnostic and vaccination failures,

thus hampering disease control measures. These results also

point out to the need for permanent viral monitoring and genetic

and antigenic characterization of the isolates throughout the

country.

Acknowledgements.-Acknowledgements.- To Dr. José Paulo G. Leite at Instituto Oswaldo Cruz (FIOCRUZ/RJ) for valuable comments and suggestions. This study was supported by FAPESP (Proc. 01/10155-4).

REFERENCES

Baker J.C. 1995. The clinical manifestation of bovine viral diarrhea infection. Bovine Viral Diarrhea Virus. Vet. Clin. North Am., Food Anim. Pract.. 11:425-446.

Baule C. 2000. Molecular characterization of Bovine Viral Diarrhoea Virus, an important pathogen of cattle. Doctoral thesis, Swedish University of Agricultural Sciences, Uppsala, p.49.

Baule C., Vuuren M. van, Lowings J.P. & Belák S. 1997. Genetic heterogeneity of bovine viral viruses isolated in Southern Africa. Virus Res. 52:205-220.

Baule C., Kulcsar G., Belak K., Albert M., Mittelholzer C., Soos T., Kucsera L. & Belak S. 2001. Pathogenesis of primary respiratory disease induced by isolates from a new genetic cluster of bovine viral diarrhea virus type I. J. Clin. Microbiol. 39(1):146-53.

Canal C.W., Strasser M., Hertig C., Masuda A. & Peterhans E.1998. Detection of antibodies to bovine viral diarrhea virus (BVDV) and characterization of genomes of BVDV from Brazil. Vet. Microbiol. 63:85-97.

Collett M.S., Larson R., Gold C., Strick D., Dennis K., Anderson D.K. & Purchio A.F. 1988. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165(1):191-199. Cortez A., Heinemann M.B., Soares R.M., Castro A.M.M.G., Leite R.C. &

Richtzenhain L.J. 2005a. Sensibilidade analítica de uma hn-nested para o vírus da Diarréia Viral Bovina em amostras clínicas experimentalmente contaminadas. VI Congresso de Buiatria, Búzios, RJ, 25-28 de maio de 2005. Meio digital. htpp://www.buiatria.org.br. Disponível em 28.05.2005.

Cortez A., Heinemann M.B., Castro A.M.M.G., Scarcelli E., Genovez M.E., Vasconcellos S.A., Alfieri A.A. & Richtzenhain L.J. 2005b. Emprego da PCR no diagnóstico direto de enfermidades causadoras de distúrbios reprodutivos em bovinos e bubalinos. Meio digital. htpp:// www.buiatria.org.br. Disponível em 28.05.2005.

Flores E.F., Weiblen R., Gil L.H.V.G., Tobias F.L., Lima M., Garcez D.C & Botton S.A. 2000. Diversidade antigênica de amostras do vírus da diar-réia viral bovina isoladas no Brasil: implicações para o diagnóstico e estratégias de imunização. Arq. Bras. Med. Vet. Zootec. 52(1):11-17. T

TT T

Table 2. Genotyping results obtained by partial sequencing of a 5’-UTR Rable 2. Genotyping results obtained by partial sequencing of a 5’-UTR Rable 2. Genotyping results obtained by partial sequencing of a 5’-UTR Rable 2. Genotyping results obtained by partial sequencing of a 5’-UTR Region inable 2. Genotyping results obtained by partial sequencing of a 5’-UTR Region inegion inegion inegion in Brazilian BVDV isolates

Brazilian BVDV isolates Brazilian BVDV isolates Brazilian BVDV isolates Brazilian BVDV isolates

Isolatesa Year b Condition/Clinical signs c Origind Subgenotypee

UFSM3f ₁₉₉₇ _{Healthy calf} _{Pelotas, RS} _1a

663 f ₂₀₀₀ _{Persistent respiratory disease} _{Tapejara, RS} _1a

1/5g ₂₀₀₂ _{Aborted Foetus} _{Muriaé, MG} _1a

216 f ₂₀₀₂ _{Persistent infection} _{Interior do RS} _1a

3/4g ₂₀₀₄ _{Respiratory disease} _{Rondonópolis, MT} _1a

303 f ₂₀₀₃ _{Aborted foetus} _{Londrina, PR} _1a

132 f ₂₀₀₄ _{Aborted foetus} _{São Martinho, RS} _1a

278 f 2004 Aborted foetus São Vicente do Sul, RS 1a

395 f - Aborted foetus Londrina, PR 1b

163 f 2000 Respiratory and digestive Arroio dos Ratos, RS 1b

390 g 2002 Diarrhea Porecatu, PR 1b

44g 1997 Aborted foetus Batatais, SP 2a

323 f ₂₀₀₄ _{Gastrointestinal} _{Porto Alegre, RS} _2a

728 f ₂₀₀₂ _{Gastrointestinal} _RS _2b

11f ₂₀₀₃ _{Gastrointestinal} _{Cruz Alta, RS} _2b

301 f ₂₀₀₃ _{Persistent infection} _{Carambeí, PR} _2b

302 f ₂₀₀₃ _{Persistent infection} _{Carambeí, PR} _2b

200 g ₂₀₀₂ _{Aborted foetus} _{São Sebastião da Grama, SP Atypical}_Pestivirus

315 g ₂₀₀₄ _{Aborted foetus} _{Tambaú, SP} _{Atypical Pestivirus}

(6)

Flores E.F., Ridpath J.F., Weiblen R., Vogel F.S.F. & Gil L.H.V.G. 2002. Phylogenetic analysis of Brazilian bovine viral diarrhea virus type 2 (BVDV-2) isolates: evidence for a subgenotype within BVDV-2. Virus Res. 87:51-60.

Flores E.F., Weiblen R., Vogel F.S.F, Roehe P.M., Alfieri A.A. & Pituco E.M. 2005. A infecção pelo Vírus da Diarréia Viral Bovina (BVDV) no Brasil: histórico, situação atual e perspectivas. Pesq. Vet. Bras. 25(3):125-134.

Fray M.D., Paton D.J. & Alenius S. 2000. The effects of bovine viral diarrhoea virus on cattle reproduction in relation to disease control. Anim. Reprod. Sci. 60/61:615-627.

Fulton R.W., Ridpath, J.f., Ore S., Confer A.W., Saliki J.T., Burge L.J. & Payton M.E. 2005. Bovine viral diahrroea virus (BVDV) subgenotypes in diagnostic laboratory accessions: Distribution of BVDV-1a, 1b, and 2a subgenotypes. Vet. Microbiol. 111:35-40.

Gil L.H.V.G. 1998. Seqüenciamento, análise filogenética e caracterização de polipeptídeos não-estruturais de amostras do Vírus da Diarréia Viral Bovina (BVDV). Dissertação de Mestrado, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul. 69p.

Gil L.H.V.G., Botton S.A., Odeon S.A., Weber L., Weiblein R., Ridpath J.F. & Flores E.F. 1998. Genotypic analysis of South America isolates of bovine viral diarrhea virus. Simpósio Internacional sobre Herpesvírus bovino (tipos 1 e 5) e Vírus da Diarréia Viral Bovina, Santa Maria, RS, p.177. (Resumo)

Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98.

Hamers C., Dehan P., Couvreur B., Letellier C., Kerkhoes P. & Pastoret P.-P. 2001. Diversity among bovine pestivirus. Vet. J. 161:112-122

Houe H. 1999. Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections. Vet. Microbiol. 64:89-107. Hueselsenbeck J.P. & Rannala B. 1987. Phylogenetic methods come of age:

testing hypothesis in evolucionary context. Science 276:227-32. Jones L.R., Zandomeni R. & Webwe L.E. 2001. Genetic typing of bovine

viral diarrhea virus isolates from Argentina. Vet. Microbiol. 81:367-375.

Jones L.R., Cigliano M.M., Zandomeni R.O. & Weber E.L. 2004. Phylogenetic analysis of bovine pestiviruses: testing the evolution of clinical symptoms. Cladistics 20(5):443-453.

Kumar S., Tamura K., Jakobsen I.B., Nei M. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17(12):1244-1245.

Letellier C., Kerkhofs P., Wellwmans G. & Vanopdenbosch E. 1999. Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5’untranslated region. Vet. Microbiol. 64:155-167.

Lindeberg A. & Houe H. 2005. Characteristics in the epidemiology of bovine viral diarrhea virus (BVDV) of relevance control. Prev. Vet. Med. 72:55-73

Meyer G., Rumenopf T. & Thiel H.-J. 1989. Molecular cloning and nucleotide sequence of the genoma of hog cholera virus. Virology 171:555-567.

Nagai M., Sato M., Nagano H., Pang H., Kong X., Murakami T., Ozawa T. & Akashi H. 1998. Nucleotide sequence homology to bovine viral diarrhea virus 2 (BVDV-2) in the 5' untranslated region of BVDVs from cattle with mucosal disease or persistent infection in Japan. Vet. Microbiol. 60(2-4):271-276.

Pellegrin C., Van den Hurk J., Lecomte J. & Tijssen P. 1994. Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities. Virology 203:260-268.

Posadas J. & Crandall K.A. 1988. Modeltest: testing the model of DNA substitution. Bioinformatics 14(9):817-18.

Ridpath J.F., Bolin S.R. & Dubovi E.J. 1994. Segregation of Bovine Viral Diarrhea Virus into genotypes. Virology 205:66-74.

Ridpath J.F. & Bolin S.R. 1995. The genomic sequence of a virulent bovine viral diarrhea virus (BVDV) from the type 2 genotype: detection of a large genomic insertion in a non-cytopathic BVDV. Virology 212 (1):39-4 6

Ridpath J.F. & Bolin S.R. 1998. Differentiation of types 1a, 1b and 2 bovine viral diarrhea virus (BVDV) by PCR. Mol. Cell. Probes 12(2):101-106.

Ridpath J.F., Neill J.D., Vilcek S., Dubovi E.J. & Carman S. 2006. Multiple outbreaks of severe acute BVDV in North America occurring between 1993 and 1995 linked to the same BVDV2 strain. Vet. Microbiol. 114(3-4):196-204.

Sandvick T., Paton D. & Lowings P.J. 1997. Detection and identification of ruminant and porcine pestivirus by nested amplification of 5’untranslated cDNA regions. J. Virol. Methods 64:43-56.

Schirrmeier H., Strebelow G., Depner K., Hoffman B. & Beer M. 2004. Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species. J. Gen. Virol. 85:3647-3652

Tajima M., Frey H., Osamu, Y., Maede Y., Moeninig V., Scholz H. & Greiser-Wilke I. 2001. Prevalence of genotypes 1 and 2 of bovine viral diarrhea virus in Lower Saxony, Germany. Virus Res. 76:31-42.

Vilcek S., Nettleton P.F., Paton D.J. & Belak S. 1997. Molecular character-ization of ovine pestiviruses. J. Gen. Virol. 78(Pt 4):725-735

Vilcek S., Paton D.J., Durkovic B., Strojny L., Ibata G., Moussa A., Loitsch A., Rossmanith W., Vega S., Scicluna M.T. & Paifi V. 2001. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch. Virol. 146(1):99-115.

Vilcek S., Durkovic B., Kolesarova M., Greiser-Wilke I. & Paton D. 2004. Genetic diversity of international bovine viral diarrhoea virus (BVDV) isolates: identification of a new BVDV-1 genetic group. Vet. Res. 35:609-615.